Antithrombin III (ATIII) plays a critical role in maintaining the fluidity of blood. Blood coagulation is mediated by a series of serine proteases. Antithrombin III is a potent inhibitor of Factors IXa, Xa (Kurachi et al., 1976).sup.1, XI (Damus et al., 1973), XIIa (Stead et al., 1976), and thrombin (Rosenberg et al., 1973). Thus, ATIII regulates clot formation both at the activation level and the thrombin level. The physiological importance of ATIII in preventing excessive coagulation is revealed by studies of individuals whose antithrombin levels are decreased due to heredity (Egeberg, 1965; Odegard et al., 1977; Van der Meer et al., 1973; and Sas et al., 1974) or acquired deficiency (Abildgaard et al., 1970; Mannucci et al., 1973; Fagerhol et al., 1970). Such persons are prone to spontaneous thrombosis and the associated risks of disseminated intravascular coagulation (DIC), cardiac infarction, cerebrovascular accident, pulmonary embolism, etc. Transfusion of patients suffering from severe bleeding disorders complicated by DIC with antithrombin III concentrates obtained by blood fractionation has suggested that such replacement therapy can restore normal hemostatic function (Schipper et al., 1978). FNT .sup.1 Reference is made to the appended bibliography which is hereby made a part hereof, the publications and other materials there correspondingly cited in more detail being incorporated herein by reference.
Human antithrombin III is a single-chain glycoprotein synthesized by the liver and found in plasma at a concentration of approximately 20 mg/dl (Collen et al., 1977). Molecular weight determinations for antithrombin III have yielded values between 54,000 and 65,000 daltons (Rosenberg and Damus, 1973; Nordenman et al., 1977; Kurachi et al., 1976), of which some 10 percent is carbohydrate (Kurachi et al., 1976). The primary amino acid structure of human antithrombin III has been almost completely determined by Petersen et al. (1979) who reported that this protein has approximately 430 amino acid residues, 4 glucosamine-based oligosaccharide side units, and 3 disulfide bridges. However, these workers were uncertain as to the precise identity of several amino acids, and moreover, did not identify the sequence and length of an amino acid stretch in the middle of the protein due to incompletely overlapped peptides.
ATIII inactivates thrombin by forming a covalent 1:1 stoichiometric complex with the protease (Rosenberg and Damus, 1973; Owen, 1975). The anticoagulant effect of antithrombin III is enhanced by heparin, which greatly increases the rate of inhibitor-protease complex formation (Abildgaard, 1968).
It was thought possible that therapeutic administration of human antithrombin III produced by genetically engineered microorganisms would also be useful in the clinical prevention and management of thromboses. Such genetic engineering methods would provide sufficient quantities of material so as to enable its clinical testing as a demonstration of the safety and efficacy prerequisites to marketing. Therefore, the task was undertaken of cloning the gene for human antithrombin III and expressing it in a host cell.